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Prepubescent children need to be prepared for puberty in an appropriate manner including all related associations and contexts. Children should learn the required knowledge before its onset—when they are in primary school. Knowledge is gained in the process of learning and represents the level of awareness. The cognitive level of knowledge represents the amount and quality of relevant information. Adolescence is a broadly defined stage of life, in which the reproductive capacity culminates and physical growth is completed. In the period of adolescence, psychological and social changes take place along with biological changes. Puberty is a significant element of sexuality education in the global dimension. The objective of the present educational research study is to identify the level of knowledge concerning the definition and significance of puberty among primary school pupils in Croatia and in the context of other selected countries (Czech Republic, Sweden, China, Spain). The data were collected by means of a knowledge achievement test. The content of the nine test items focused on the following: concept of puberty; definition of puberty; puberty age range; knowledge about physical changes in boys and girls; knowledge about other changes that puberty induces; significance of puberty in human life. To identify any statistically significant differences in the pupils’ responses by countries, the non-parametric Kruskal-Wallis test was used. The results suggest that prepubescents do not have the right knowledge about puberty and do not consider relevant associations and contexts in a comprehensive way. The incomplete knowledge suggests that prepubescents do not think about the significance of puberty in a comprehensive perspective but rather in various combinations of the biological, psychological and social areas.